The present invention concerns new sulphated compounds of hyaluronic acid and derivatives thereof having anticoagulant and antithrombotic activities and processes for their preparation. Said compounds are useful in the preparation of pharmaceutical compositions and biomaterials and in the production of coatings for biomedical objects.
Heparin is the sulphated glycosaminoglycan with the greatest biological activity. Its antithrombotic and anticoagulant properties are well known. Indeed, it is used in treatment for cardiovascular pathologies where there is a risk of thrombosis, and it has contributed notably to the successful outcome of open-heart surgery. The structure of heparin is not altogether known.
Commercial heparin comprises a range of 21 different kinds (Nader et al., 1974, Biochem. Biophys. Res. Commun. 57:488), with a molecular weight of between 3,000 and 37,500 Da, and with varying anticoagulant activity.
Heparin""s anticoagulant activity depends on its structural characteristics, for example, on the degree of sulphation, on the degree of dissociation, on the sequence of the COOxe2x80x94 and SO3xe2x80x94 groups, on the shape and size of the molecule. These factors are important to the formation of the ion bonds responsible for heparin""s biological activity (Stivala et al., 1967, Arch. Biochem. Biophys. 122:40).
Because of the high density of its negative charge, heparin has a strong affinity for cations and its activity is pH-dependent. In particular, the N-sulphated group of its glucosamine residue plays a fundamental role in the interaction with the factors regulating the coagulative processes.
A significant reduction in the N-sulphated groups drastically reduces its anticoagulant and antithrombotic activities.
Many natural polysaccharides have been sulphated in order to obtain heparin-like products (Hoffman et al., 1982, Carbohydrate Res. 2:115; Kindness et al., 1980, Brit. J. Pharmac. 69:675; Horton et al., 1973, Carbohydrate Res. 30:349; Okada et al., 1979, Makromol. Chem. 180:813; Kikuchi et al., 1979, Nippon Kagaku Kaishi 1:127; Manzac et al., 1981, Proc. Third M.I.S.A.O. 5:504). Moreover, sulphuric, carboxy or sulphonated groups have been attached to synthetic polymers such as polystyrene (Kanmaugue et al., 1985, Biomaterials, 6:297) and polyurethanes (Ito et al., 1992, Biomaterials, 13:131).
However, the anticoagulant activity of these materials is much lower than that of heparin and depends on the type of substituent, the type of bond, the degree of substitution and the sequence.
Lastly, some chemical reactions for the sulphation of polysaccharides are known (WO 88/00211; EP 0340628; Carbohydrate Research, 158, 183-190, 1986) but no derivatives have ever been obtained which present, besides the chemical-physical characteristics peculiar to polysaccharides, any new characteristics such as anticoagulant activity.
In the international patent application, Publication No. WO 95/25751, a process is described for the non-selective sulphation of hyaluronic acid and the derivatives thereof to obtain compounds with an antithrombotic activity.
Their ability to inactivate thrombin is due to the formation of electrostatic interactions depending on the charge density, which increases according to the degree of sulphation, while heparin""s activity is the consequence of a direct interaction with antithrombin III (T. W. Barrowcliffe et al., Journal of Pharmaceutical and Biomedical Analysis, Vol. 7, No. 2, pages 217-226, 1989; Peter D. J. Grootenhuiis et al., J. Am. Chem. Soc., 113, 2743-2747, 1991).
Heparin is widely used, although it does present side effects such as haemorrhagic effects, which prevent its being used too freely or without medical guidance.
Moreover, because of its chemical-physical characteristics, heparin cannot be used as a biomaterial but simply as a coating for other materials, and in sufficiently small quantities to avoid its causing localized bleeding.
Lastly, by acting directly on the coagulation factors, the anticoagulant action of heparin begins very rapidly and its duration, albeit dose-dependent, is generally similarly brief. These drawbacks limit its applicability in certain surgical fields such as cardiovascular surgery involving the implantation of devices requiring an absolute absence of thrombogenicity for a given length of time.
The present invention is directed to novel sulphated compounds of hyaluronic acid and the derivatives thereof, optionally salified, with an anticoagulant and antithrombotic activity, wherein the glucosamines are partially N-sulphated or partially N-sulphated and partially or totally O-sulphated in position 6, for the preparation of pharmaceutical formulations, biocompatible and bioabsorbable biomaterials with an anticoagulant activity and for the coating of biomedical objects and processes for their preparation.
The present invention also provides for a chemical process using a well-characterized starting product such as hyaluronic acid, which process allows for the selective sulphation of the amino group of glucosamine or the hydroxy group in the position 6, to thus obtain new sulphated derivatives of hyaluronic acid with an unaltered range of molecular weights and with an anticoagulant activity similar to that of heparin.
While the ability of hypersulphated polysaccharides to inactivate thrombin is due to the formation of electrostatic interactions depending on the charge density, which increases according to the degree of sulphation, the N-sulphated derivatives provided in the present invention appear to act on the coagulation factors by means of a specific an mechanism similar to that of heparin.
Further advantages of the present invention are represented by the improved chemical-physical characteristics of the N-sulphated derivatives compared to those of the hyper-sulphated derivatives, making them suitable for the preparation of biomaterials for use in the fields of biomedicine and health care and in the pharmaceutical field.
Moreover, the possibility of obtaining a compound with an anticoagulant and non-thrombogenic activity by means of selective sulphation of just the amino groups and hydroxy groups in position 6, notably reduces the costs of the process compared to the preparation of hyaluronic acid with all the hydroxy groups homogeneously sulphated.